Urease activity assay

Peers, S., MiUigan, A., and Harrison, P. (2000). Assay optimization and regulation of urease activity in two marine diatoms. J. Phycol. 36, 523-528. 

Detection limits in EIA are ultimately determined by how low one can measure the label s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, /8-galactosi-dase, peroxidase, - and glucose oxidase. This is due to the high mo-... 

Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. 

Equipment. All potentiometric measurements were taken on a Coming Model 12 research pH meter in conjunction with a Heath-Schlum-berger Model SR-255B strip chart recorder. An Orion Model 95-10 ammonia gas sensing electrode was used for all assays of urease activity. Potentiometric activity measurements were made at 25° in a 10-ml glass thermostatted cell. 

Laboratory tests such as urease activity, protein dispersibility index (PDI), nitrogen solubility index (NSI), thiamine, and water absorption have been found valuable in monitoring daily production for protein quality. But biological chick and/or rat assays are the only reliable means currently available for predetermining the nutritional value of whole soybean protein they must be conducted periodically to verify results of chemical tests (31). If whole soybeans are to be used in a mixture containing 20% or more soybean meal, 5% or more urea, and 20% or more molasses, or an equivalent mixture, and exposed to hot, humid storage conditions, it is advisable that the urease activity of the whole soybeans not exceed 0.12 increase in pH (31). Extruded or roasted soybeans properly treated for cattle to increase bypass protein should have urease values of less than 0.05 pH rise. A urease rise of 0.05-0.20 is an indication of proper treatment for swine and poultry. 

General Assay Urease Activity in Problem Soils. Tu (14) examined urease activity in clay loam soils incubated with various pesticides. He observed increased levels of microbial urease activity with carbofuran- and ethoprop-amended soils (Table I). These results suggest that activity levels of specific indigenous soil enzymes may be influenced by certain pesticides applied to... 

Immobilization of Urease on Sweetzyme Pellets For co-immobilization of urease on the Sweetzyme pellets, 500 ml of 1 g/1 urease solution and 2 g of Sweetzyme pellets was added to a 1-1 beaker [35]. The beaker was left on the benchtop at room temperature for 24 h. The pellets were separated from the solution by decanting and gravity filtration and dried on a paper towel at room temperature for 24 h or until dry. Co-immobilized pellets were stored at 4 °C until use. Activity of immobilized urease was measured at pH 7.5 and 25°C using a standard assay procedure that measures the rate of ammonia liberation [43]. The urease activities obtained with our immobilization procedure were in the range of 550-577 U/g pellets, where a unit liberates 1 pmol of ammonia per minute under the assay conditions. 

MAY P.B. and DOUGLAS L.A. 1976. Assay for soil urease activity. Plant and Soil, 301-305. 

Vol.l., Eds. McLaren A.D. and Peterson G.H. Marcel Dekker, New York. SKUJINS J.J. and McLAREN A.D. 1969. Assay of urease activity using C-urea in stored, geologically preserved, and in irradiated soils. Soil Biology and Biochemistry, 1, 89-99. 

TABATABAI M.A. and BREMNER J.M. 1972. Assay of urease activity in soil. Soil Biology and Biochemistry, 4, 479-487. 

The activity of enzymes in the film was estimated in the following way In order to test the activity of urease, we utilized a calorimetric assay based on urea hydrolysis the enzymatic reaction was followed at 590 nm, the suitable wavelength for bromcresol purple (Chandler 1982). Urea concentration was 1.67 ts 10 M. 

Groves and Teng (1992) investigated the effect of compactional pressure on biologically active proteinaceous enzymes such as a-amylase, P-glucuronidase, lipase, and urease. Assaying the activity of these enzymes before and after the compaction... 

Workers in several laboratories have noted that the activity of urease appears to increase upon standing at room temperature. Until this is understood assay procedures cannot be assumed to yield precise values. 

Ammonium carbamate is formed in citrate or Tris buffer. It is a 480-kDa protein with a pH optimum of 6.0. The enzyme is very specific for urea or hydroxyurea. It is inhibited by heavy metals, while it is stabilized by 1 X 10 M EDTA. The specific activity of urease is very high (10 000 U/mg), but the potential detection sensitivity is lost because the reaction products are difficult to detect. Urease conjugates because of free thiols are not particularly stable. Urease is important in noninstrumented assays because activity can be visualized via pH-sensitive dyes. 

Figure 6 shows a typical calibration curve obtained for the inhibition of binding of a urease-cAMP conjugate to anti-cAMP antibody as determined by the ammonia electrode. One hundred percent of activity refers to blank tubes, which had rates of 11-12 mV/min in the absence of cAMP. Selectivity of the assay over structurally similar cGMP is also shown in Fig. 6. It takes approximately 1000 times more cGMP than...

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